Effects of HCM Missense Mutations in the M Domain of Cardiac Myosin Binding Protein C on Calcium Sensitivity of Force and Rate in Rat Trabeculae

Cardiac myosin-binding protein C (cMyBP-C) is a functional sarcomeric protein that regulates contractility in response to contractile demand, and many mutations in cMyBP-C lead to hypertrophic cardiomyopathy (HCM). To gain insight into the effects of disease-causing cMyBP-C missense mutations on contractile function, we expressed the pathogenic W792R mutation (substitution of a highly conserved tryptophan residue by an arginine residue at position 792) in mouse cardiomyocytes lacking endogenous cMyBP-C and studied the functional effects using three-dimensional engineered cardiac tissue constructs (mECTs). Based on complete conservation of tryptophan at this location in fibronectin type II (FnIII) domains, we hypothesized that the W792R mutation affects folding of the C6 FnIII domain, destabilizing the mutant protein. Adenoviral transduction of wild-type (WT) and W792R cDNA achieved equivalent mRNA transcript abundance, but not equivalent protein levels, with W792R compared with WT controls. mECTs expressing W792R demonstrated abnormal contractile kinetics compared with WT mECTs that were nearly identical to cMyBP-C-deficient mECTs. We studied whether common pathways of protein degradation were responsible for the rapid degradation of W792R cMyBP-C. Inhibition of both ubiquitin-proteasome and lysosomal degradation pathways failed to increase full-length mutant protein abundance to WT equivalence, suggesting rapid cytosolic degradation. Bacterial expression of WT and W792R protein fragments demonstrated decreased mutant stability with altered thermal denaturation and increased susceptibility to trypsin digestion. These data suggest that the W792R mutation destabilizes the C6 FnIII domain of cMyBP-C, resulting in decreased full-length protein expression. This study highlights the vulnerability of FnIII-like domains to mutations that alter domain stability and further indicates that missense mutations in cMyBP-C can cause disease through a mechanism of haploinsufficiency. NEW & NOTEWORTHY This study is one of the first to describe a disease mechanism for a missense mutation in cardiac myosin-binding protein C linked to hypertrophic cardiomyopathy. The mutation decreases stability of the fibronectin type III domain and results in substantially reduced mutant protein expression dissonant to transcript abundance.

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Biological variation of cardiac myosin-binding protein C in healthy individuals

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2021-0306 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Bashir Alaour ◽

Torbjørn Omland ◽

Janniche Torsvik ◽

Thomas E. Kaier ◽

Marit S. Sylte ◽

...

Keyword(s):

Risk Stratification ◽

Protein C ◽

Myocardial Injury ◽

Binding Protein ◽

Biological Variation ◽

Cardiac Myosin ◽

Analytical Quality ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

Abstract Objectives Cardiac myosin-binding protein C (cMyC) is a novel biomarker of myocardial injury, with a promising role in the triage and risk stratification of patients presenting with acute cardiac disease. In this study, we assess the weekly biological variation of cMyC, to examine its potential in monitoring chronic myocardial injury, and to suggest analytical quality specification for routine use of the test in clinical practice. Methods Thirty healthy volunteers were included. Non-fasting samples were obtained once a week for ten consecutive weeks. Samples were tested in duplicate on the Erenna® platform by EMD Millipore Corporation. Outlying measurements and subjects were identified and excluded systematically, and homogeneity of analytical and within-subject variances was achieved before calculating the biological variability (CVI and CVG), reference change values (RCV) and index of individuality (II). Results Mean age was 38 (range, 21–64) years, and 16 participants were women (53%). The biological variation, RCV and II with 95% confidence interval (CI) were: CVA (%) 19.5 (17.8–21.6), CVI (%) 17.8 (14.8–21.0), CVG (%) 66.9 (50.4–109.9), RCV (%) 106.7 (96.6–120.1)/−51.6 (−54.6 to −49.1) and II 0.42 (0.29–0.56). There was a trend for women to have lower CVG. The calculated RCVs were comparable between genders. Conclusions cMyC exhibits acceptable RCV and low II suggesting that it could be suitable for disease monitoring, risk stratification and prognostication if measured serially. Analytical quality specifications based on biological variation are similar to those for cardiac troponin and should be achievable at clinically relevant concentrations.

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Cardiac myosin‐binding protein C as a candidate biomarker in heart failure: rational but not revolutionary

European Journal of Heart Failure ◽

10.1002/ejhf.2126 ◽

2021 ◽

Author(s):

A. Mark Richards

Keyword(s):

Heart Failure ◽

Protein C ◽

Binding Protein ◽

Cardiac Myosin ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

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Cardiac Myosin-Binding Protein C Modulates the Tuning of the Molecular Motor in the Heart

Biophysical Journal ◽

10.1529/biophysj.107.127787 ◽

2008 ◽

Vol 95 (2) ◽

pp. 720-728 ◽

Cited By ~ 21

Author(s):

Yves Lecarpentier ◽

Nicolas Vignier ◽

Patricia Oliviero ◽

Aziz Guellich ◽

Lucie Carrier ◽

...

Keyword(s):

Protein C ◽

Molecular Motor ◽

Binding Protein ◽

Cardiac Myosin ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

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Phosphorylation of cardiac myosin binding protein C releases myosin heads from the surface of cardiac thick filaments

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614020114 ◽

2017 ◽

Vol 114 (8) ◽

pp. E1355-E1364 ◽

Cited By ~ 35

Author(s):

Robert W. Kensler ◽

Roger Craig ◽

Richard L. Moss

Keyword(s):

Protein C ◽

Structural Changes ◽

Binding Protein ◽

Cardiac Contraction ◽

Cardiac Myosin ◽

Cross Bridge ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Thick Filaments ◽

Myosin Binding

Cardiac myosin binding protein C (cMyBP-C) has a key regulatory role in cardiac contraction, but the mechanism by which changes in phosphorylation of cMyBP-C accelerate cross-bridge kinetics remains unknown. In this study, we isolated thick filaments from the hearts of mice in which the three serine residues (Ser273, Ser282, and Ser302) that are phosphorylated by protein kinase A in the m-domain of cMyBP-C were replaced by either alanine or aspartic acid, mimicking the fully nonphosphorylated and the fully phosphorylated state of cMyBP-C, respectively. We found that thick filaments from the cMyBP-C phospho-deficient hearts had highly ordered cross-bridge arrays, whereas the filaments from the cMyBP-C phospho-mimetic hearts showed a strong tendency toward disorder. Our results support the hypothesis that dephosphorylation of cMyBP-C promotes or stabilizes the relaxed/superrelaxed quasi-helical ordering of the myosin heads on the filament surface, whereas phosphorylation weakens this stabilization and binding of the heads to the backbone. Such structural changes would modulate the probability of myosin binding to actin and could help explain the acceleration of cross-bridge interactions with actin when cMyBP-C is phosphorylated because of, for example, activation of β1-adrenergic receptors in myocardium.

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S ‐glutathiolation impairs phosphoregulation and function of cardiac myosin‐binding protein C in human heart failure

The FASEB Journal ◽

10.1096/fj.201500048 ◽

2016 ◽

Vol 30 (5) ◽

pp. 1849-1864 ◽

Cited By ~ 13

Author(s):

Konstantina Stathopoulou ◽

Ilka Wittig ◽

Juliana Heidler ◽

Angelika Piasecki ◽

Florian Richter ◽

...

Keyword(s):

Heart Failure ◽

Protein C ◽

Human Heart ◽

Binding Protein ◽

Cardiac Myosin ◽

Human Heart Failure ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding ◽

And Function

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Calcium Handling Derangement is Associated with Conditional Cardiac Myosin Binding Protein C Knock Out

Biophysical Journal ◽

10.1016/j.bpj.2011.11.1244 ◽

2012 ◽

Vol 102 (3) ◽

pp. 226a-227a

Author(s):

Erin M. Capes ◽

Randall Loaiza ◽

Peter P. Chen ◽

Daniel P. Fitzsimons ◽

Hector H. Valdivia ◽

...

Keyword(s):

Protein C ◽

Binding Protein ◽

Calcium Handling ◽

Cardiac Myosin ◽

Knock Out ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

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Cardiac myosin binding protein-C phosphorylation accelerates β-cardiac myosin detachment rate in mouse myocardium

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00673.2020 ◽

2021 ◽

Author(s):

Bertrand C.W. Tanner ◽

Michael J. Previs ◽

Yuan Wang ◽

Jeffrey Robbins ◽

Bradley M. Palmer

Keyword(s):

Protein C ◽

Binding Protein ◽

Cardiac Contraction ◽

Power Stroke ◽

Cardiac Myosin ◽

Force Development ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Detachment Rate ◽

Myosin Binding

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament protein that modulates cardiac contraction-relaxation through its phosphorylation. Phosphorylation of cMyBP-C and ablation of cMyBP-C have been shown to increase the rate of MgADP release in the acto-myosin crossbridge cycle in the intact sarcomere. The influence of cMyBP-C on Pi-dependent myosin kinetics has not yet been examined. We investigated the effect of cMyBP-C and its phosphorylation on myosin kinetics in demembranated papillary muscle strips bearing the b-cardiac myosin isoform from non-transgenic (NTGβ) and transgenic mice lacking cMyBP-C (t/tβ). We used quick stretch and stochastic length-perturbation analysis to characterize rates of myosin detachment and force development over 0-12 mM Pi. Protein kinase-A (PKA) treatment was applied to half the strips to probe the effect of cMyBP-C phosphorylation on Pi-sensitivity of myosin kinetics. Increasing Pi increased myosin crossbridge detachment rate similarly for muscles with and without cMyBP-C, although these rates were higher in muscle without cMyBP-C. Treating myocardial strips with PKA accelerated detachment rate when cMyBP-C was present over all Pi, but not when cMyBP-C was absent. The rate of force development increased with Pi in all muscles. However, Pi sensitivity of the rate force development was reduced when cMyBP-C was present vs. absent, suggesting that cMyBP-C inhibits Pi-dependent reversal of the power stroke or stabilizes crossbridge attachment to enhance the probability of completing the power stroke. PKA treatment reduces the role for cMyBP-C to slow myosin detachment and thus effectively accelerates b-myosin detachment in the intact myofilament lattice.

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